Our long-term goal is to develop genetic approaches that could be used for the treatment of mitochondrial disorders associated with mitochondrial DNA (mtDNA) mutations. Although some of the approaches tested in the past showed some promise, they are inefficient and unlikely to reach clinical trials. We propose to develop approaches that are more efficient in improving oxidative phosphorylation in cells harboring heteroplasmic mtDNA mutations (i.e. a mixture of wild-type and mutated mtDNA). The approach is based on the mtDNA heteroplasmy shift concept, where, by genetic manipulation, levels of the wild-type genome are increased in relation to the mutated genome. In the previous funding period we showed that mitochondria-targeted specific endonucleases are powerful tools to achieve mtDNA heteroplasmy shift. The present proposal will continue to develop this approach. We will deliver mitochondria targeted restriction endonucleases systemically to muscle of a mouse model of mtDNA heteroplasmy and expect to see a generalized shift in mtDNA heteroplasmy in skeletal muscle. To expand the use of mitochondria nucleases, we will create novel zinc finger protein nucleases that can target common mtDNA mutations associated with mitochondrial disorders. When this approach moves into the clinical arena, it will be important to avoid secondary effects with potential clinical relevance, such as transient mtDNA depletion. To this end, we will test a model whereby the levels of mtDNA are increased (per cell) before the action of the specific mitochondrial restriction endonuclease. This should minimize a potential bioenergetic crisis associated with partial mtDNA depletion.